This invention relates to modification of the apoptotic response of target cells, for instance target cells in a subject. More specifically, it relates to apoptosis-modifying fusion proteins with at least two domains, one of which targets the fusion protein to a target cell, and another of which modifies an apoptotic response of the target cell.
Tissue and cell homeostasis in multicellular organisms is largely influenced by apoptosis, the phenomenon of programmed cell death by which an intra- or extra-cellular trigger causes a cell to activate a biochemical xe2x80x9csuicidexe2x80x9d pathway. Morphological indicia of apoptosis include membrane blebbing, chromatin condensation and fragmentation, and formation of apoptotic bodies, all of which take place relatively early in the process of programmed cell death. Degradation of genomic DNA during apoptosis results in formation of characteristic, nucleosome sized DNA fragments; this degradation produces a diagnostic xcx9c180 bp laddering pattern when analyzed by gel electrophoresis. A later step in the apoptotic process is degradation of the plasma membrane, rendering apoptotic cells leaky to various dyes (e.g., trypan blue and propidium iodide). Apoptotic cells are usually engulfed and destroyed early in the death process; thus, apoptosis tends not to be associated with inflammation caused by cytoplasm leakage, as is found in necrosis.
Various in vivo triggers can induce apoptosis; the paradigmatic trigger is a shortage of one or more necessary growth factors. Apoptosis plays a significant role in development of the neural system (reviewed in Cowan et al., Science 225:1258-1265, 1984; Davies, Development 101:185-208, 1987; Oppenheim, Annu. Rev. Neurosci. 14:453-501, 1991) and lymphoid system (reviewed in Blackman et al., Science 248:1335-1341, 1990; Rothenberg, Adv. Immunol. 51:85-214, 1992) of vertebrates. System development occurs through selective apoptotic extinction of certain cell populations.
In spite of much study, the molecular mechanisms of apoptosis are not fully elucidated. It does appear, however, that different apoptosis inducers may trigger different apoptotic pathways. For instance, certain pathways are transcription-dependent, in that apoptosis requires the synthesis of new proteins after stimulation by, for instance, withdrawal of growth factors. Staurosporine, a non-specific kinase inhibiter, in contrast, stimulates a transcription-independent pathway. Transcription dependent and independent pathways appear to share downstream components, including the ICE family of proteases (caspases). See Rubin, British Med. Bulle., 53:617-631, 1997, for a review of apoptosis in neurons; More general reviews include Ashkenazi and Dixit, Science 281:1305-1308; Thornberry and Lazebnik, Science 281:1312-1316; and Adams and Cory, Science 281:1322-1326.
Apoptosis is recognized as a gene-directed event, controlled by a complex set of interacting gene products that inhibit or enhance apoptosis (Williams and Smith, Cell 74:777-779, 1993; reviewed in White, Genes Dev. 10:1-15, 1996). Extensive effort is currently underway to identify and characterize the genes involved in this process. The first protein characterized as influencing apoptosis was Bcl-2 (Cleary et al., Cell 47:19-28, 1986; Tsujimoto and Croce, Proc. Natl. Acad Sci. USA 83:5214-5218, 1986). Since its discovery, several Bcl-2-related proteins (the Bcl-2 family of proteins) have been identified as being involved in regulation of apoptosis (White, Genes Dev. 10:1-15, 1996; Yang et al., Cell 80:285-291, 1995). One such is Bcl-x, which is expressed in two different forms, long (Bcl-xL) and short (Bcl-xS) (Boise et al., Cell 74:597-608, 1993).
Bcl-xL and certain other members of the Bcl-2 family are, like Bcl-2 itself, powerful inhibitors of cell death (the xe2x80x9canti-deathxe2x80x9d Bcl-2 family members). Genetic overexpression of Bcl-2 has been shown to block apoptosis in the nervous system of transgenic mice (Chen et al., Nature 385:434-439, 1997; Henkart, Immunity 4:195-201, 1996; Lippincott-Schwartz et al., Cell 67:601-616, 1991; Hunziker et al., Cell 67:617-627, 1991; Krajewski et al., Cancer Research 53:4701-4714, 1993; Martinou et al., Neuron 13:1017-1030, 1994).
Other members of the Bcl-2 protein family, including Bcl-xS, Bad and Bax, are potent enhancers of apoptosis and therefore toxic to cells (xe2x80x9cpro-deathxe2x80x9d Bcl-2 family members). Though the mechanism of apoptosis induction by these proteins remains unknown, it has been suggested that Bad binding to Bcl-xL may promote cell death (Yang et al., Cell 80:285-291, 1995; Zha et al., J Biol. Chem 272:24101-24104, 1997) and that phosphorylation of Bad may prevent its binding to Bcl-xL, thereby blocking cell death (Zha et al., J Biol. Chem. 272:24101-24104, 1997; Zha et al., Cell 87:619-628, 1996).
In addition to its involvement in neuronal and lymphoid system development and overall cell population homeostasis, apoptosis also plays a substantial role in cell death that occurs in conjunction with various disease and injury conditions. For instance, apoptosis is involved in the damage caused by neurodegenerative disorders, including Alziheimer""s disease (Barinaga, Science 281:1303-1304), Huntington""s disease, and spinal-muscular atrophy. There is also a substantial apoptotic component to the neuronal damage caused during stroke episodes (reviewed in Rubin, British Med. Bulle., 53(3):617-631, 1997; and Barinaga, Science 281:1302-1303), and transient ischemic neuronal injury, as in spinal cord injury. It would be of great benefit to prevent undesired apoptosis in various disease and injury situations.
Treatment with standard apoptosis inhibitory molecules, for instance peptide-type caspase inhibitors (e.g., DEVD-type), though useful for laboratory experiments where microinjection can be employed, has proven unsatisfactory for clinical work due to low membrane permeability of these inhibitors. Transfection of cells with various native proteins, including members of the Bcl-2 family of regulatory proteins, has dual disadvantages. First, transfection is usually not cell-specific, and thus may disrupt apoptotic processes non-specifically in all cells. Second, transfection tends to provide long term alterations in the apoptotic process, in that once a transgene is integrated and functional in the genome of target cells, it may be difficult to turn off. Especially in instances of stroke episodes or transient ischemic neuronal injury, it would be more advantageous to be able to apply apoptosis regulation for short periods of time. Therefore, there is still a strong need to develop pharmaceutical agents that overcome these disadvantages.
Cancer and other hyper-proliferative cell conditions can be viewed as inappropriate escape from appropriate cell death. As such, it would be advantageous to be able to enhance apoptosis in certain of these cells to stop unregulated or undesired growth. Various attempts have been made to selectively eliminate cancerous cells through the use of targeted immunotoxins (genetic or biochemical fusions between a toxic molecule, for instance a bacterial toxin, and a targeting domain derived, typically from an antibody molecule).
One bacterial toxin that has been employed in attempts to kill cancerous cells is diphtheria toxin (DT). Diphtheria toxin has three structurally and functionally distinct domains: (1) a cell surface receptor binding domain (DTR), (2) a translocation domain (DTT) that allows passage of the active domain across the cell membrane, and (3) the A (enzymatically active) chain that, upon delivery to a cell, ADP-ribosylates elongation factor 2 and thereby inactivates translation. Altering the receptor specificity of the diphtheria toxin has been used to generate toxins that may selectively kill cancer cells in vitro (Thorpe et al., Nature 271:752-755, 1978) and in man (Laske et al., Nature Medicine 3:1362-1368, 1997). Promising though they might have seemed, these and similar hybrid immunotoxins have proven to be substantially less effective at targeted cell death than the toxins from which they were generated. This is perhaps due to difficulties in translocation of the fusion protein into the target cell (Columbatti et al., J. Biol. Chem. 261:3030-3035, 1986). In addition, in vivo results have been particularly poor using such hybrid constructs (Fulton et al., Fed. Proc. 461:1507, 1987).
It is to biological molecules that overcome deficiencies in the prior art that the present invention is directed.
Disclosed herein are apoptosis-modifying fusion proteins constructed by fusing a protein, or an apoptosis-modifying fragment or variant thereof, from the Bcl-2 protein family with a cell-binding, targeting domain such as one derived from a bacterial toxin. Using this approach, apoptosis-modifying fusion proteins can be delivered effectively throughout the body and targeted to select tissues and cells. In certain embodiments, fusing various cell-binding domains to Bcl-2 family proteins (such as Bcl-xL or Bad) allows targeting to specific subsets of cells in vivo, permitting treatment and/or prevention of the cell-death related consequences of various diseases and injuries. The delivery of other Bcl-2 homologues to the cell permits regulation of cell viability either positively (using anti-death Bcl-2 family members), or negatively (using pro-death members of the Bcl-2 family).
The apoptosis-modifying fusion proteins disclosed herein have specifiable cell-targeting and apoptosis-modifying activities. Thus, they may be used clinically to treat various disease and injury conditions, through inhibition or enhancement of an apoptotic cellular response. For instance, apoptosis-inhibiting fusion proteins are beneficial to minimize or prevent apoptotic damage that can be caused by neurodegenerative disorders (e.g., Alzheimer""s disease, Huntington""s disease, spinal-muscular atrophy), stroke episodes, and transient ischemic neuronal injury (e.g., spinal cord injury). The apoptosis-enhancing fusion proteins n can be used to inhibit cell growth, for instance uncontrolled cellular proliferation.
Accordingly, a first embodiment is a functional apoptosis-modifying fusion protein capable of binding a target cell, having a first domain capable of modifying apoptosis in the target cell, and a second domain capable of specifically targeting the fusion protein to the target cell. This fusion protein further integrates into or otherwise crosses a cellular membrane of the target cell upon binding to that cell.
Certain embodiments will also include a linker between these two domains. This linker will usually be at least 5 amino acids long, for example between 5 and 100 amino acids in length, and may for instance include the amino acid sequence shown in SEQ ID NO: 6. Appropriate linkers may be 6, 7, or 8 amino acids in length, and so forth, including linkers of about 10, 20, 30, 40 or 50 amino acids long.
The apoptosis modifying fusion proteins may also include a third domain from one of the two original proteins, or from a third protein. This third domain may improve the fusion protein""s ability to be integrated into or otherwise cross a cellular membrane of the target cell. An example of such a third domain is the translocation region (domain or sub-domain) of diphtheria toxin.
Target cells for the fusion proteins disclosed herein include, but are not limited to, neurons, lymphocytes, stem cells, epithelial cells, cancer cells, neoplasm cells, and others, including other hyper-proliferative cells. The target cell chosen will depend on what disease or injury condition the fusion protein is intended to treat.
Receptor-binding domains may be derived from various cell-type specific binding proteins, including for instance bacterial toxins (e.g., diphtheria toxin or anthrax toxin), growth factors (e.g., epidermal growth factor), monoclonal antibodies, or single-chain antibodies derived from antibody genes. Further, variants or fragments of such proteins may also be used, where these fragments or variants maintain the ability to target the fusion protein to the appropriate target cell.
Further specific embodiments employ essentially the entire Bcl-xL protein as the apoptosis-modifying domain of the fusion protein, or variants or fragments thereof that maintain the ability to inhibit apoptosis in a target cell to which the protein is exposed. Examples of such proteins are fusion proteins made of the Bcl-xL protein, functionally linked to the diphtheria toxin receptor binding domain through a peptide linker of about six amino acids. One such protein is Bcl-xL-DTR, which consists of Bcl-xL and DTR, without the translocation domain of diphtheria toxin. The nucleotide sequence of this fusion protein is shown in SEQ ID NO: 1, and the corresponding amino acid sequence in SEQ ID NOs: 1 and 2.
Another such example is LFn-Bcl-xL, which includes the amino terminal portion (residues 1-255) of mature anthrax lethal factor (LF), coupled to residues 1-209 of Bcl-xL. The nucleotide sequence of this fusion protein is shown in SEQ ID NO: 7, and the corresponding amino acid sequence in SEQ ID NOs: 7 and 8.
Also encompassed are fusion proteins wherein the apoptosis-modifying domain is an apoptosis-enhancing domain. Such domains include the various pro-death members of the Bcl-2 family of proteins, for instance Bad, and variants or fragments thereof that enhance apoptosis in a target cell. A specific appropriate variant of the Bad protein has an amino acid other than serine at amino acid position 112 and/or position 136, to provide constitutively reduced phosphorylation.
Thus, one specific embodiment is a functional apoptosis-enhancing fusion protein capable of binding a target cell, comprising the Bad protein and the diphtheria toxin translocation and receptor binding domains, functionally linked to each other. The Bad protein of this embodiment can also contain a mutation(s) at position 112 and/or 136 to change the serine residue to some other amino acid, to reduce phosphorylation of the protein. One such protein is Bad-DTTR; the nucleotide sequence of this protein is shown in SEQ ID NO: 3, and the corresponding amino acid sequence in SEQ ID NOs: 3 and 4.
Also disclosed herein are nucleic acid molecules encoding apoptosis-modifying fusion proteins, for instance the nucleic acid sequences in SEQ ID NOs: 1, 3, and 7, and nucleic acid sequences having at least 90% sequence identity to these sequences, for instance those encoding for proteins containing one or more conservative amino acid substitutions. Other nucleic acid sequences may have 95% or 98% sequence identity with SEQ ID NO: 1, 3, or 7. Also encompassed are recombinant nucleic acid molecules in which such a nucleic acid sequence is operably linked to a promoter, vectors containing such a molecule, and transgenic cells comprising such a molecule.
Methods also are provided for producing functional recombinant apoptosis-modifying fusion proteins capable of binding to a target cell, integrating into or otherwise translocating across the cell membrane, and modifying an apoptotic response of the target cell. Such a protein can be produced in a prokaryotic or eukaryotic cell, for instance by transforming or transfecting such a cell with a recombinant nucleic acid molecule comprising a sequence which encodes a disclosed bispecific fusion protein. Appropriate cukaryotic cells include yeast, algae, plant or animal cells. Such transformed cells can then be cultured under conditions that cause production of the fusion protein, which is then recovered through protein purification means. The protein can include a molecular tag, such as a six histidine (hexa-his) tag, to facilitate its recovery.
Protein analogs, derivatives, or mimetics of the disclosed proteins, which retain the ability to target to appropriate target cells and modify apoptosis in those cells, are also encompassed in embodiments.
Compositions containing these apoptosis modifying fusion proteins, and analogs, derivatives, or mimetics of these proteins, are further aspects of this disclosure. Such compositions may further contain a pharmaceutically acceptable carrier, various other medical or therapeutic agents, and/or additional apoptosis modifying substances.
Methods for modifying apoptosis in a target cell are also encompassed, wherein a sufficient amount of a fusion protein of the current disclosure to modify apoptosis in the target cell is contacted with a target cell. Modification of apoptosis can be by either inhibition or enhancement of an apoptotic response of the target cell. The fusion protein can be administered to the target cell in the form of a pharmaceutical composition, and can further be administered with various medical or therapeutic agents, and/or additional apoptosis modifying substances. Such agents may include, for instance, chemotherapeutic, anti-inflammatory, anti-viral, and antibiotic agents.
Bcl-xL-DTR, LFn-Bcl-xL, or related fusion proteins can be used to inhibit apoptosis in a target cell by contacting the target cell with an amount of this protein sufficient to inhibit apoptosis. Alternatively, Bad-DTTR or related fusion proteins can be used to enhance apoptosis in a target cell by contacting the target cell with an amount of this protein sufficient to enhance apoptosis.
A specific aspect disclosed herein is the method of reducing apoptosis in a subject after transient ischemic neuronal injury, for instance a spinal cord injury, comprising administering to the subject a therapeutically effective amount of an apoptosis-inhibiting protein according to this disclosure. Examples of such fusion proteins include Bcl-xL-DTR and LFn-Bcl-xL. These proteins can be administered in the form of a pharmaceutical composition, and can be co-administered with various medical or therapeutic agents, and/or additional apoptosis modifying substances.